Colonization, spread and persistence of Salmonella (Typhimurium, Infantis and Reading) in internal organs of broilers

Transfer of Salmonella to internal organs of broilers over a 35 d grow-out period was evaluated. A total of 360 one-day old chicks were placed in 18 floor pens of 3 groups with 6 replicate pens each. On d 0, broilers were orally challenged with a cocktail of Salmonella (equal population of marked serovars; nalidixic acid-resistant S. Typhimurium, rifampicin-resistant S. Infantis, and kanamycin-resistant S. Reading) to have 3 groups: L (low; ∼2 log CFU/bird); M (medium; ∼5 log CFU/bird); and H (High; ∼8 log CFU/bird). On d 2, 7 and 35, 4 birds/pen were euthanized and ceca, liver, and spleen samples were collected aseptically. Gizzard samples (4/pen) were collected on d 35. The concentration of Salmonella in liver and spleen were transformed to binary outcomes (positive and negative) and fitted in glm function of R using cecal Salmonella concentrations (log CFU/g) and inoculation doses (L, M, and H) as inputs. On d 2, H group showed greater (P ≤ 0.05) cecal colonization of all 3 serovars compared to L and M groups. However, M group showed greater (P ≤ 0.05) colonization of all 3 serovars in the liver and spleen compared to L group. Salmonella colonization increased linearly in the ceca and quadratically in the liver and spleen with increasing challenge dose (P ≤ 0.05). On d 35, L group had greater (P ≤ 0.05) S. Infantis colonization in the ceca and liver compared to M and H groups (P ≤ 0.05). Moreover, within each group on d 35, the concentration of S. Reading was greater than those of S. Typhimurium and S. Infantis for all 3 doses in the ceca and high dose in the liver and gizzard (P ≤ 0.05). Salmonella colonization diminished in the ceca, liver, and spleen during grow-out from d 0 to d 35 (P ≤ 0.05). On d 35, birds challenged with different doses of Salmonella cocktail showed a similar total Salmonella spp. population in the ceca (ca. 3.14 log CFU/g), liver (ca. 0.54 log CFU/g), spleen (ca. 0.31 log CFU/g), and gizzard (ca. 0.42 log CFU/g). Estimates from the fitted logistic model showed that one log CFU/g increase in cecal Salmonella concentration will result in an increase in relative risk of liver and spleen being Salmonella-positive by 4.02 and 3.40 times (P ≤ 0.01), respectively. Broilers from H or M group had a lower risk (28 and 23%) of being Salmonella-positive in the liver compared to the L group when the cecal Salmonella concentration is the same (P ≤ 0.05). Oral challenge of broilers with Salmonella spp. with various doses resulted in linear or quadratic increases in Salmonella colonization in the internal organs during early age and these populations decreased during grow-out (d 35). This research can provide guidance on practices to effectively mitigate the risk of Salmonella from chicken parts and enhance public health.


INTRODUCTION
Salmonellosis is a major foodborne illness and has been linked to consumption of poultry products including meat and eggs (Antunes et al., 2016).Interagency Food Safety Analytics Collaboration (IFSAC) estimated over 23% of salmonellosis can be contributed to consumption of chicken and turkey products (IFSAC, 2021).Adoption of strategies at preharvest to reduce both Salmonella prevalence and concentration is required to achieve a greater reduction in Salmonella loads in broiler meat.
Among all serovars, Salmonella Typhimurium and S. Enteritidis were the most studied serovars in broilers as they are responsible for most human cases (Finstad et al., 2012;Lamas et al., 2018, Punchihewage-Don et al., 2022).S. Infantis is an emerging serovar that is increasingly causing greater human illness in the U.S. and Europe (Alba et al., 2020;Tack et al., 2020).The incidence of S. Infantis has increased since 2016 and accounted for 17.7% (30/170) of Salmonella-positive samples in 2020 in the U.S. (Siceloff et al., 2022).The European Food Safety Authority (EFSA, 2018) identified S. Infantis as the most frequent serovar in broiler flocks (45.6%) and broiler meat (50.6%) and showed an increasing prevalence in breeder flocks.S. Reading is rarely associated with human illness from chicken products.Recent outbreaks linked to consumption of turkey products have made S. Reading a prominent serovar for public health (Hassan et al., 2019).Few studies have evaluated colonization and translocation of S. Infantis and S. Reading to internal organs during grow-out of broilers.
Salmonella challenge models (seeder birds or oral/cloacal gavage) are often used to induce gastrointestinal (GI) tract colonization to evaluate the efficacy of dietary interventions on mitigating Salmonella colonization in broilers.In such studies, chicks are commonly challenged with Salmonella at a high dose of 6 to 8 log CFU/mL through the oral route to ensure a successful colonization, and cecal Salmonella concentration was enumerated through early stages of the birds' life to evaluate the efficacy of tested product (Shanmugasundaram et al., 2019;Yadav et al., 2022).Regardless of the differences in challenge models used in broiler research, limited literature on the effect of challenge dose on colonization of Salmonella in the GI tract and translocation to other internal parts is available, particularly simulating commercial production practices such as raising birds in floor pens.
The majority of studies on Salmonella spread to broiler parts reported prevalence as a main indicator of movement and persistence in liver or spleen (Adhikari et al., 2020;Choi et al., 2022).However, reporting Salmonella prevalence provides limited information on colonization and translocation to internal organs as the food safety risk of Salmonella in those products is potentially related to their concentrations, and risk mitigation through preparation and cooking practices (Harris et al., 2003).Reports on Salmonella concentration in broiler edible organs such as liver and gizzard especially at the preharvest stage are limited, and this information can be valuable to develop risk mitigation recommendations for consumers of these products.The objective of this study was to quantify the transfer and persistence of Salmonella spp. to various internal organs of broilers that were orally inoculated with different doses of a Salmonella cocktail containing the serovars Typhimurium, Infantis and Reading.

MATERIALS AND METHODS
The experimental protocol was reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee.

Salmonella Challenge and Birds Husbandry
Three Salmonella serovars, nalidixic acid-resistant S. Typhimurium (ST) obtained from the U.S. National Poultry Research Center (NPRC-USDA, Athens, GA), rifampicin-resistant strain of S. Infantis (5 isolates; SI) and kanamycin-resistant S. Reading (5 isolates; SR) obtained from the Food Safety and Inspection service (FSIS) were used in this study.Cultures of the bacteria were maintained as frozen stocks.The Salmonella serovar cocktail for the challenge was prepared as previously described (Yadav et al., 2022).Briefly, 1 d prior to challenge, 5 colonies of ST isolated from BG Sulfa agar (BGS; Difco, Sparks, MD) containing 200 ppm nalidixic acid were used to inoculate 10 mL of tryptic soy broth (TSB; Remel, Lenexa, KS) also supplemented with 200 ppm nalidixic acid, the cultures were then incubated for 18 to 24 h at 35 § 1°C.Preparation of SI and SR inoculum followed similar procedure except for the corresponding antibiotic marker (rifampicin and kanamycin) supplemented to BGS and TSB.Five tubes of each strain were pooled into three 50 mL conical tubes and centrifuged at 7,838 £ g for 10 min at 4°C (Model 5430R, Eppendorf North America, Enfield, CT) to collect the cells.The individual pellets of each serovar were re-suspended with 10 mL 0.1% buffered peptone water (BPW; Difco TM, Sparks, MD) and centrifuged again.The supernatant was removed to wash away the antibiotics, the pellet resuspended in 5 mL of BPW, and the OD values of suspension were read at a wavelength 400 nm using spectrometer (Genesys 10S UV-VIS, Thermo Scientific, Waltham, MA).The inoculum was serially diluted using PW to prepare the target inoculum dose: L (»2 log CFU/mL), M (»5 log CFU/mL) and H (»8 log CFU/mL).
A total of 360 Cobb male broilers were obtained from a local hatchery (Cobb Vantress, Cleveland, GA) and randomly allocated to the 3 treatment groups based on the 3 doses of Salmonella cocktail inoculum (1 mL/bird containing equal population of marked serovars ST, SI, and SR).Birds were reared in floor pens with fresh litter in an environmentally controlled room at the UGA Poultry Research Center.The room temperature and lighting schedule were set following the Cobb Broiler Management Guide (Cobb-Vantress, 2018b).A corn and soybean mealbased diet meeting or exceeding Cobb Nutrition Guide requirements (Cobb-Vantress, 2018a) was fed to all treatment groups.Birds had ad libitum access to water and feed throughout the 35-d grow-out period.

Sample collection and Salmonella Enumeration
On d 2, 7, and 35, internal organs (ceca, liver, and spleen) of 4 birds from each pen were aseptically collected for Salmonella enumeration.The cecal sample weights ranged from 0.5 to 1.5 g, 1 to 3 g and 10 to 20 g on d 2, 7, and 35, respectively.Similarly, the liver and spleen samples weighed 1 to 3 g, 4 to 8 g, and 30 to 45 g and 0.1 to 0.3 g, 1 to 3 g and 2 to 4 g, respectively on d 2, 7, and 35 d.Additionally, the gizzard was aseptically everted to expose the inner layer using a scalpel, the contents were removed on d 35 and the gizzard was used for microbiological enumeration.All samples were placed in sterile filter bags (Nasco, Weber Scientific, Hamilton Township, NJ) and weighed.
Chilled BPW (10 mL for samples [ceca, liver, and spleen] from d 2 and 7; 40 mL for samples from d 35) was added into each sample and stomached (Neutec Group Inc., Farmingdale, NY) for 1 min.Chilled BPW (40 mL) was added to each gizzard sample and stomached as described.Serial dilutions were prepared in sterile 0.1% BPW, and appropriate dilutions were plated on duplicate BGS agar plates supplemented with nalidixic acid, rifampicin, or kanamycin to enumerate the ST, SI, and SR, respectively.The plates were incubated for 24 h at 35 § 1°C, typical colonies were enumerated and reported as log 10 CFU/g.Simultaneously, an aliquot of the sample (3 mL) was added to BAX MP medium (Hygiena, Camarillo, CA) supplemented with nalidixic acid, rifampicin, or kanamycin (57 mL for ceca sample and 3 mL for liver and spleen samples) and incubated for 24 h at 35 § 1°C.Salmonella prevalence was determined using BAX System SalQuant (Hygiena, Camarillo, CA) for samples below the detection limit.

Statistical Analysis
All enumeration data were log-transformed and analyzed using one-way ANOVA using the GLM procedure of SAS OnDemand for Academics (SAS Institute, Cary, NC).Separation of means was performed using Tukey's range test at a = 0.05.Polynomial orthogonal contrast was used to determine the linear and quadratic effects of the challenge dose.
Additionally, logistic regression was performed in R (Version 4.0.2,RCore Team, 2021) using glm function with family = binomial argument.Salmonella prevalence was used as the response, and the cecal Salmonella concentration (log CFU/g) and treatment (L, M, and H) were used to determine the correlation.The Salmonella concentration (log CFU/g) in liver and spleen was transformed into prevalence (positive and negative).Individual birds were used as the data input (n = 3 treatments £ 6 replicates £ 4 birds £ 3 time points = 216 total).Figures were generated as change of probability of liver/spleen being tested positive (0−100%) against cecal Salmonella concentration (log CFU/g) using ggplot2 package.

Colonization and Translocation of Salmonella in Broiler Internal Organs
Salmonella population in the L, M, and H challenge doses were 2.82, 5.13, and 8.39 log CFU/mL, respectively.Salmonella concentration in the ceca increased linearly (P ≤ 0.01) with the challenge dose, from 2 to 8 log CFU/bird, 2 d post-inoculation (dpi) for all 3 serovars and the pooled count (Table 1).Day-old chicks challenged with all 3 doses were able to establish colonization of Salmonella in the ceca with 6.75, 7.47, and 8.50 log CFU/g for L, M, and H groups at 2 dpi, respectively.Pooled Salmonella concentration of 3 serovars in the liver and spleen from M dose group birds were higher than L dose group (P ≤ 0.05).The concentrations of 3 individual Salmonella serovars and their pooled counts in the liver and spleen showed a quadratic increase (P ≤ 0.01) as the challenge dose increased.
The pooled counts of 3 serovars in the ceca, liver, and spleen at 7 dpi increased quadratically (P ≤ 0.05) with increasing challenge dose, reaching a peak for Med dose birds (Table 2).Chicks challenged with a L dose of Salmonella showed greater SI Rif colonization (6.24 log CFU/g) compared to ST (5.38 log CFU/g) in the ceca at 7 dpi (P ≤ 0.05).Broilers from all 3 treatment groups had a similar Salmonella population in the ceca (approx.3 log CFU/g) at 35 Table 1.Salmonella colonization and persistence (log CFU/g) in the ceca and internal organ invasion (liver and spleen) in day-old chicks during grow out as affected by different serovars (Typhimurium, Infantis, and Reading) and challenge dose (approx.2, 5, and 8 log CFU/chick).

SALMONELLA COLONIZATION AND SPREAD IN BROILERS
dpi.Salmonella concentrations in the liver, spleen and gizzard at 35 dpi were also similar for all 3 treatments with <1 log CFU/g (Table 3).

Persistence of Salmonella in Broiler Internal Organs
Salmonella persisted in the ceca of the birds throughout the 35 d grow-out period, regardless of the challenge dose (Figure 1).Salmonella concertation in ceca decreased rapidly from 8.50 to 6.83 at 2 and 7 dpi, with a subsequent gradual decline to 3.45 log CFU/g by 35 dpi in H dose challenge group.A similar trend was observed for Salmonella concentration in the ceca of birds from L and M groups from ca. 7 log CFU/g at 2 dpi to ca. 3 log CFU/g at 35 dpi.
Irrespective of the serovar or the challenge dose, Salmonella was able to colonize the ceca and invade the internal organs (spleen and liver).Salmonella populations increased quadratically in the liver and spleen during the early infection period of 2 and 7 dpi (approx. 2 −4 log CFU/g) followed by a gradual decrease (<1 log CFU/g) up to 35 dpi (Figures 2 and 3).Different from Salmonella cecal colonization and persistence, M dose group resulted in higher Salmonella concentrations at 2 and 7 dpi (approx.3−4 log CFU/g) compared to the other 2 doses (approx.2−3 log CFU/g), although the concentration decayed over time to <1 log CFU/g for birds in all 3 treatment groups.Additionally, the difference of Salmonella population (log CFU/organ) in the spleen and liver at the same sampling time were less than 0.3 log CFU/g, which may suggest that the translocation is non-specific in broiler internal organs.
Salmonella population of all 3 serovars in broiler ceca and internal organs were similar (P > 0.05) at 2 and 7 dpi.However, SR showed greater persistence in the ceca (all 3 challenge dose groups) compared to ST and SI and  Same superscripts ( abc ) within the same column indicate no significant differences (P > 0.05); same superscripts ( xy ) within the first 3 row (3 serovars of S. Typhimurium, S. Infantis, and S. Reading.) indicate no significant differences (P > 0.05).numerically higher counts in the liver, spleen and gizzard at 35 dpi.

Logistic Regression
Results of logistic regression were presented in Figures 4 and 5.The estimates and statistics of the logistic regression model is shown in Table 4.As the increase of cecal Salmonella concentration from 0 to 9 log CFU/ g, the probability of liver or spleen to be tested positive for Salmonella increased from 0 to almost 100%.More specifically, for every 1-log CFU/g increase in cecal Salmonella load, the relative risk of liver and spleen being Salmonella positive increased by 4.02 and 3.40 times.Moreover, broilers from H and M group had a lower risk (28 and 23%, P≤0.05) of liver being positive for Salmonella compared to the birds in L group for the same cecal concentration.

DISCUSSION
Oral and/or cloacal challenges were the more common and effective routes for colonization of Salmonella in the gastrointestinal tract, with a faster colonization and invasion of the internal organs achieved via the cloacal route (Bailey et al., 2005).We used the oral challenge as  it replicates the normal oral-fecal route of transmission of the chicks in a poultry house, either through the feed, water, litter, pests, or other sources in poultry production and also to achieve a consistent colonization of the 1-day-old chicks.
Oral challenge of Salmonella (7−9 log CFU/bird) in young broilers has been commonly used to establish the infection model that is used to evaluate nutritional strategies on controlling Salmonella in broiler live production (Knap et al., 2011;Shanmugasundaram et al., 2019).The authors reported that Salmonella challenge resulted in colonization of the chicks followed by translocation to other body tissues and persisted throughout the 5-wk grow-out period in the current study.The low pH of the upper gastrointestinal tract is a contributing factor for the higher level of Salmonella required for colonizing young chicks (Bailey et al., 2005).However, even the L dose challenge (ca. 2 log CFU/bird) resulted in successful colonization and invasion of the liver and spleen in the present study, probably due to the immature gastrointestinal tract of newly hatched chicks.A higher Salmonella challenge dose in broilers resulted in a linear increase in the cecal colonization (Bailey et al., 2005).Additionally, a challenge dose of 2-8 log CFU/bird of Salmonella resulted in a high population of Salmonella (6-8 log CFU/g) in the ceca during the first week.reported that a challenge dose of 3 log CFU/bird of S. Typhimurium in 1-day-old chicks resulted in a 5 log CFU/g in ceca and ileum by d 8 dpi.Salmonella concentration decreased gradually during the grow-out, with ca. 3 log CFU/g Salmonella recovered from cecal samples at 35 dpi.Stern (2008) reported cecal Salmonella concentration decayed rapidly from 6 to 1 log CFU/g during 28 d period in birds challenged from 4-8 log CFU/bird of Salmonella.
During in vivo Salmonella challenge trials, regardless of oral challenge or aerosol exposure, Salmonella cecal population decreased gradually over time after the challenge (Marcq et al., 2011;Pal et al., 2021).Bjerrum et al. (2003) also reported a rapid decrease in Salmonella population in the ileum and ceca following a challenge with 7.2 log CFU of S. Typhimurium on day of hatch, over a 35-d period in isolators.With the natural coprophagic behavior, raising broilers in floor pens with access to contaminated litter may lead to continuous colonization of the gastrointestinal tract and subsequent spread to their internal organs.Yadav et al. (2022) reported similar Salmonella concentration in broiler ceca was <1 log CFU/g subsequent to 21 d of grow-out when the chicks/birds were grown in battery cages (limiting reexposure to Salmonella from the environmental sources), using the same ST serovar and challenged with 7 log CFU/bird on d 1.However, Bohorquez (2007) reported that broilers reared in cages or on litter had a higher concentration of Salmonella in the ceca (ca. 3 log CFU/g) on d 42, in chicks orally challenged on d 3 with nonmarked strains of Salmonella serovars.The differences of Salmonella colonization in the ceca of birds reared in cages and floor pens may be due to the marker strains used for challenge or differences in the serovars/strains of Salmonella used for the challenge.The use of marked strains minimizes the impact of background microbiota in the chicken gut on enumeration and allows a more precise tracking of Salmonella population (Bailey et al., 2005).The findings of the present study also suggest that when resources allow, Salmonella challenge of 1day-old chicks followed by rearing on floor pens may be a more reliable model for evaluating the effectiveness of dietary interventions during broiler production/growout.This represents real life scenario, where the 1-dayold chicks are exposed to Salmonella in the hatchery as well as in the poultry house environment after placement.Meanwhile, an oral Salmonella challenge concentration at 2 to 5 log CFU/bird is adequate to colonize the 1-day-old chicks under those rearing conditions.
In addition to the gradual decrease in Salmonella population in the ceca, broilers challenged with 3 doses eventually stabilize at comparable Salmonella concentration in  the ceca (ca. 3 log CFU/g) at 35 d of age.Under commercial production, exposure of 1-day-old chicks to Salmonella is probably limited at a low or medium Salmonella concentrations as the litter has been reported to contain low levels of Salmonella, with a mean population of 1.70 log MPN/g (range of <1.0 to 3.6 log MPN/g; Gutierrez et al., 2020).Regardless of the Salmonella concentration in the environmental sources such as water, feed or litter, Salmonella exposure can result in exponential population increase in in the GI tract, followed by persistent colonization in the ceca and invasion of internal organs (spleen and liver).It is possible that the birds entering the processing plant may carry up to 3-log of Salmonella populations in the ceca and antimicrobial interventions designed at processing facilities should be able to address and reduce these Salmonella populations (resulting from cross contamination of the carcass during processing) to assure the safety of poultry meat.Despite several limitations in the logistic regression currently used such as that data points were acquired from a mixture of 2, 7, and 35 d-old birds, and the cecal concentrations were only at ca. 3 log CFU/g at 35 d of age.The current results suggested that a higher concentration of Salmonella in ceca is associated with a higher probability that other internal organs (liver and spleen) tare positive for Salmonella.However, a lower risk was observed in birds challenged with H and M doses of Salmonella.A possible explanation is that the immune system plays a role in controlling Salmonella translocation.Previous studies have shown that when a Salmonella challenge was subclinical and asymptotic, intestinal integrity was not affected after 6 d post inoculation (Yadav et al., 2022).Gut permeability increased 72 h after broilers were challenged with Salmonella Enteritidis (Prado-Rebolledo et al., 2017).Choi et al. (2022) reported broilers challenged with Salmonella exhibited gut leakage at 14 d but recovered by 21 d of age.The differences in gut permeability suggests that intestinal immune regulation is related to the infection time as well as the maturation of the immune system.Additional research is necessary to elucidate immune response of Salmonella challenged birds and possibly use it as an intervention method to reduce Salmonella incidence in broilers prior to harvest.The findings of the present study indicate a possible link between the Salmonella concentration at pre-harvest (in litter, cecal droppings, or cecal contents) and its prevalence in other internal organs, necessitating further investigation.Spread of Salmonella to internal organs (liver and spleen) requires crossing of the microorganisms through the GI tract and subsequent systemic translocation and colonization (Adhikari et al., 2020;Shi et al., 2021;Choi et al., 2022).Salmonella translocation to internal organs such as the liver and spleen is often reported as prevalence, rather than the population.Microbial risk assessments for the safety of chicken parts such as liver, gizzard and others consumed as food require quantitative data to evaluate the risk and identify risk reduction strategies.Data on the Salmonella concentration in liver and spleen in market age birds (broilers) are lacking.
Higher Salmonella challenge dose resulted in a linear increase in cecal Salmonella population.However, the concentration of Salmonella recovered from liver and spleen quadratically increased with increasing challenge dose and stabilized at M dose group.The differences in the Salmonella concentrations observed in the ceca versus the liver and spleen may be due to the immune response of the bird.Immunity to Salmonella infection in young chicks is mainly from the maternal antibodies and the activity of both innate and adaptive immune system, with natural killer cells, intraepithelial T cells and macrophages as critical factors (Meijerink et al., 2021).Spleen, as the secondary immune organ not only functions as blood filtration, but also is a key immune organ for systemic antigen sampling and mounting appropriate humoral and/or cell-mediated immune responses (Smith and Hunt, 2004).Hamad et al. (2017) reported that host-defense peptides play a key role in the innate immunity components with direct antimicrobial activities and immunomodulatory properties in poultry species.Exposing the chicks to a high Salmonella population (8 log CFU/g) as in the H dose challenge possibly could trigger a more acute immune response and upregulation of related immune genes such as avian beta-defensins and cathelicidins.This results in fewer Salmonella cells disseminated systematically to the liver and spleen in comparison to the lower challenge doses (L and M).Additional studies are necessary to evaluate the relationship between the challenge dose and the immune responses of the birds.
Foodborne outbreaks and illnesses from consumption of inadequately cooked, contaminated chicken livers and gizzards have been reported over the past 20 yr (Abdalrahman and Fakhr, 2015;Lanier et al., 2018).Jung et al. (2019) reported an overall Salmonella prevalence of 59.4% with concentration from 6.4 to 254 MPN/g on chicken liver products in the U.S. Oscar (2021) reported a median Salmonella concentration of 1.8 log MPN/58 g (average weight of one chicken liver) at a retailer in Maryland.The Salmonella detected in products in the market may be due to the microorganisms present in the product (inside tissues) resulting from colonization of the GI tract and subsequent spread to other tissues (e. g., liver) or on the surface resulting from cross contamination during processing (Byrd et al., 2002;Berrang et al., 2019).In the present study, Salmonella concentrations in the liver, spleen, and gizzard decreased to <1 log CFU/g in market age birds (35 d).Irrespective of the challenge dose, all 3 Salmonella serovars were able to colonize the ceca and cause subsequent systemic translocation to other tissues (liver and liver) as well as in the gizzard at market age.Nagel et al. (2013) stated that interventions at pre-harvest stage that can reduce Salmonella populations to ca. 2-log in the ceca could successfully reduce the risk of Salmonella in edible parts (e. g., gizzard and liver) and consequent risk of foodborne illness from consumption of these parts.The results from the current study corroborated that Salmonella in the chicken gut can translocate and persist in broiler internal organs during live production.Therefore, incorporating effective antimicrobial interventions on those edible organs to address both the internal as external (surface) Salmonella contamination becomes necessary at the post-harvest stage to minimize the Salmonella contamination.
Salmonella serotypes Typhimurium and Infantis were linked to foodborne illness outbreaks and illnesses from consumption of chicken meat, while S. Reading was recognized as a significant issue with turkey products including ground meat and burgers (Hassan et al., 2019;CDC, 2019).Tyson et al. (2021) reported rapid spread of an emergent Salmonella Infantis clone with a large megaplasmid (pESI) containing the extended-spectrum beta-lactamase gene, which may be associated to the sharp increase in the illness linked to Infantis recently.Results from the present study illustrated that S. Reading can colonize chicken gut and could be a potential threat to broiler production as well, being a dominant strain over S. Typhimurium and S. Infantis, since it persisted in broiler ceca at 35 d.Additionally, S. Reading spread to the internal organs (liver, spleen, and gizzard) was observed, although foodborne illness outbreaks of S.
Reading have not been linked to chicken meat.It is interesting that a higher population S. Reading was observed in the ceca at 35 d compared to Typhimurium and Infantis.There is a need for further research to evaluate this comparative advantage of these serovars in the chicken gut.Measures to minimize the sources of Salmonella for the birds and subsequent colonization and spread to other organs as well as measures to reduce the Salmonella populations in the birds at market age should be implemented at pre-harvest stage during broiler production.

CONCLUSIONS
A low challenge dose of Salmonella (2 log CFU/bird) was adequate to colonize the GI tract of 1-day old chicks and subsequent spread to internal organs.Regardless of the Salmonella challenge dose and the concentrations subsequent to the challenge, similar populations of Salmonella were observed in the liver, spleen, and gizzard at market age (35 d) of these broilers.Salmonella Reading showed a greater persistence and population in the chicken ceca at market age, although the populations in the liver, spleen and gizzard were similar to other serovars (Typhimurium and Infantis).Meanwhile, reducing the cecal Salmonella load reduced the prevalence in liver and spleen.The Salmonella populations in chicken organ meats (liver and gizzard) at market age (35 d) were low, and conventional cooking practices used for preparation of these chicken parts should be adequate to eliminate the risk of bacterial survival and minimize occurrence of foodborne illness outbreaks.

Figure 1 .
Figure 1.Salmonella population (log CFU/g; pooled counts of 3 serovars S. Typhimurium, S. Infantis, and S. Reading) in ceca of broilers during 35 d grow-out that were orally challenged with a 3 serovar cocktail of different challenge dose (approx.2, 5 and 8 log CFU/chick).

Figure 2 .
Figure 2. Salmonella population (log CFU/g; pooled counts of 3 serovars S. Typhimurium, S. Infantis, and S. Reading) in liver of broilers during 35 d grow-out that were orally challenged with a 3 serovar cocktail of different challenge dose (approx.2, 5 and 8 log CFU/chick).

Figure 3 .
Figure 3. Salmonella population (log CFU/g; pooled counts of 3 serovars S. Typhimurium, S. Infantis, and S. Reading) in spleen of broilers during 35 d grow-out that were orally challenged with a 3 serovar cocktail of different challenge dose (approx.2, 5 and 8 log CFU/chick).

Figure 4 .
Figure 4. Probability of liver Salmonella positivity in broilers that were orally challenged with a 3-serovar cocktail of Salmonella (S.Typhimurium, S. Infantis, and S. Reading) of different challenge dose (ca.2, 5 and 8 log CFU/chick).

Figure 5 .
Figure 5. Probability of spleen Salmonella positivity in broilers that were orally challenged with a 3-serovar cocktail of Salmonella (S.Typhimurium, S. Infantis, and S. Reading) of different challenge dose (ca.2, 5 and 8 log CFU/chick).

Table 2 .
Salmonella colonization and persistence (log CFU/g) in the ceca and internal organ invasion (liver and spleen) on d 7 of grow out as affected by different serovars (Typhimurium, Infantis, and Reading) and challenge dose (approx.2, 5, and 8 log CFU/chick).Same superscripts ( abc ) within the same column indicate no significant differences (P > 0.05); same superscripts ( xy ) within the first 3 row (3 serovars of S. Typhimurium, S. Infantis, and S. Reading.) indicate no significant differences (P > 0.05).

Table 3 .
Salmonella colonization and persistence (log CFU/g) in the ceca and internal organ invasion (liver and spleen) on d 35 of grow out as affected by different serovars (Typhimurium, Infantis, and Reading) and challenge dose (approx.2, 5 and 8 log CFU/chick).

Table 4 .
Correlation of cecal Salmonella population and Salmonella-positivity of liver and spleen in broilers challenged with various doses of Salmonella (ca.2, 5, and 8 log CFU/chick) over 35 d period.